What is COVID-19?
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019, causing a pandemic of acute respiratory disease worldwide.1,2 This novel variant displays extensive transmission capability relative to the preceding SARS-CoV, enabling it to trigger the global pandemic of coronavirus disease 2019 (COVID-19).3
Coronaviruses encompass a diverse group of viruses that are transmissible in humans and animals.1 They were first recognized in the 1930s due to respiratory disease in poultry, following subsequent isolation and identification of infectious bronchitis viruses of chickens.3 Other pioneering work led to the identification of alphacoronavirus, and murine hepatitis virus (MHV), a form of betacoronavirus. In humans, the first coronaviruses were discovered in the 1960s.3
Two highly pathogenic coronaviruses with zoonotic origin emerged in 2002 and 2012:1 SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV).1 The latter was identified as betacoronavirus, originating in bats, which emerged as a zoonotic agent.3 These emergent coronaviruses in humans can cause fatal respiratory illness, rendering them a new public health concern of the 21st century.1 While the SARS-CoV was contained relatively rapidly, its impact resulted in a brief period of accelerated coronavirus discovery.3 Since the 2012 MERS-CoV outbreak, outbreaks at low levels have re-emerged.3
Most human coronaviruses are considered to be of bat origin; however, lineages of beta coronaviruses which include MHV, an important pathogenesis model, appear to have a rodent reservoir.3 Other forms of coronavirus, including gammacoronaviruses and a newly identified fourth genus, the deltacoronavirus, appear to have avian origin.3
Typical symptoms of COVID-19 are fever, dry cough, and fatigue and, in more severe cases, dyspnea.1 Many infections, in particular in children and young adults, are asymptomatic, whereas older adults and/or those with comorbidities possess a higher risk of severe disease, respiratory failure, and death.1 The incubation period is ~5 days, and severe disease usually develops ~8 days after symptom onset, and critical disease and death occur at ~16 days.1
How does the coronavirus work?
Coronaviruses are large, enveloped, single-stranded RNA viruses found in humans and other mammals.2 SARS-CoV-2 has a diameter of 60 nm to 140 nm and distinctive spikes, ranging from 9 nm to 12 nm, giving the virions the appearance of a solar corona.2 All of the highly pathogenic coronaviruses (CoVs), including SARS-CoV-2, belong to the betacoronavirus genus, group 2.4 The SARS-CoV-2 genome sequence shares ~80% sequence identity with SARS-CoV and ~50% with MERS-CoV.4
Its genome comprises:
- 14 open reading frames (ORFs):4
- Two-thirds of which encode 16 non-structural proteins (nsp 1–16) that make up the replicase complex
- One-third encode nine accessory proteins and four structural proteins: Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N), of which spike mediates SARS-CoV entry into host cells
The S gene of SARS-CoV-2 shares <75% nucleotide identity with SARS-CoV, demonstrating marked variability.4 The spike proteins possess a receptor-binding domain (RBD) that mediates direct contact with angiotensin-converting enzyme 2 (ACE2), a cellular receptor, and an S1/S2 polybasic cleavage site that is proteolytically cleaved by: 4
- Transmembrane protease serine 2 (TMPRSS2), which facilitates viral entry at the cell membrane surface
- Cellular cathepsin L, which activates SARS-CoV-2 spike in endosomes, and in cells that lack TMPRSS2, providing a compensatory means of entry via endosomal compartments
When the viral genome is released into the host cytosol, ORF1a and ORF1b are translated into viral replicase proteins.4 These are subsequently cleaved into individual nonstructural proteins (nsps) (via host and viral proteases: PLpro). These form the RNA-dependent RNA polymerase (nsp12 derived from ORF1b).4 Here, the replicase components rearrange the endoplasmic reticulum (ER) into double-membrane vesicles (DMVs) that facilitate viral replication of genomic and subgenomic RNAs (sgRNA); the latter are translated into accessory and viral structural proteins that facilitate virus particle formation.4
The SARS-CoV and SARS-CoV-2 lifecycle begins by envelope spike protein binding to its cognate receptor, ACE2. Entry into the host cell depends on: (i) surface TMPRSS2-mediated cleavage of the S1/S2 site ; and/or (ii) endolysosomal cathepsin L-mediated virus-cell membrane fusion, at the cell surface and endosomal membranes, respectively. Following entry via these mechanisms, the RNA genome is released into the cytosol, for translation into the replicase proteins (ORF1a/b). A virus-encoded protease cleaves the polyproteins (pp1a and pp1b) into individual replicase complex nsps (including RdRp). DMVs derived from the ER are the site of replication. The incoming positive-strand genome is the template for full-length negative-strand RNA and sgRNA. The translated sgRNA produces structural and accessory proteins (N, S, M, and E) that are assembled within the ERGIC for virion assembly. Finally, subsequent positive-sense RNA genomes form the genome of these synthesized virions, which are subsequently secreted from the plasma membrane.
Tropism of SARS-CoV-2
During the SARS epidemic, patients often presented with respiratory-like illnesses which subsequently progressed to severe pneumonia.4 This suggests that the lung is the primary tropism of SARS-CoV-2.4 As with SARS-CoV, SARS-CoV-2 finds the same entry receptor, ACE2.4 However, key mutations in the RBD of the spike protein result in additional contacts with ACE2, correlating with a greater binding affinity and increased infectivity.4 The predominant SARS-CoV-2 variant worldwide carries a D614G mutation that is absent from SARS-CoV, and is more infectious due to improved human-to-human transmission efficiency.4 The pathogenesis of severe COVID-19 is thought to be linked to mechanisms other than infectivity as the D614G variant does not correlate with disease severity.4
Alveolar epithelial cells, vascular endothelial cells, and alveolar macrophages are the first cells targeted upon infection due to their expression of ACE2.4 However, ACE2 is expressed at low levels relative to extrapulmonary tissue, suggesting permissiveness of these cells to infection is dependent on additional cell-intrinsic factors which may include:4–8
- Expression of TMPRSS2 as in the presence of undetectable amounts of ACE2, viral entry is permitted5
- mRNA expression of cellular genes, including endosomal sorting complex required for transport (ESCRT) machinery gene members as these are related to a pro-SARS-CoV-2 lifecycle and are higher in a small population of human type II alveolar cells with abundant ACE26
- Upregulation of ACE2 expression by the type I and type II interferons7
- Unique insertion of RRAR at the S1/S2 cleavage sites, which can be precleaved by furin, reducing the dependence of SARS-CoV-2 on target cell proteases (TMPRSS2/cathepsin L) for entry8
As a result of SARS-CoV-2 high affinity for ACE2, it can efficiently infect nasopharyngeal and oropharyngeal tissue which is expressed in human nasal and oral tissues.4 Human coronaviruses can cause enteric infections with variability in their pathogenicity.4 ACE2 and TMPRSS2 are expressed abundantly within mammalian intestinal tracts; consequently, gastrointestinal illness has been frequently reported in COVID-19 patients.4
Transmission of SARS-CoV-2
Human CoVs are transmitted primarily through respiratory droplets, but aerosol, direct contact with contaminated surfaces, and fecal-oral transmission were also reported during the SARS epidemic.4 Direct transmission by respiratory droplets is reinforced by productive SARS-CoV-2 replication in both the upper respiratory tract (URT) and lower respiratory tract (LRT).4
The basic reproduction number (R0) is difficult to determine as many asymptomatic infections cannot be accurately accounted for at this stage.1 However, an estimated R0 of 2.5 (ranging from 1.8 to 3.6) has been proposed for SARS-CoV-2 recently, compared with 2.0–3.0 for SARS-CoV.9
The signs and symptoms of COVID-19
COVID-19 manifestations in humans affect multiple systems in the body with varying degrees of onset and severity.4 All age groups are susceptible to infection, and the median age of infection is ~50 years.1 However, there is a clear differentiation in disease severity with regards to age. In general, older men (>60 years) with comorbidities are the most susceptible to developing severe respiratory disease that requires hospitalization or may cause death.1 By contrast, young people and children predominantly present with mild disease (non-pneumonia or mild pneumonia) or are asymptomatic.1
COVID-19 is considered to have two critical elements:1,10
- Uncontrolled SARS-CoV-2 replication which occurs in the earlier stage of the disease
- Overreactive inflammatory response (cytokine storms) in the later stage of the disease
Based on a growing understanding of the underlying pathophysiology of COVID-19 and recent clinical results, it has been proposed that effective treatment of the disease will require addressing both viral load and the inflammatory response.10
Often, manifestations in both the URT and LRT are the most noticeable if a patient is symptomatic.4 This occurs in addition to systemic symptoms, such as chills, fever, malaise, and fatigue, that are the most frequently reported regardless of the severity of disease.4
The most common symptoms are fever, fatigue, and dry cough.1 Less common symptoms include sputum production, headache, hemoptysis, diarrhea, anorexia, sore throat, chest pain, chills, and nausea and vomiting.1 Self-reported olfactory and taste disorders were also reported by patients in Italy.1
The pulmonary manifestations of COVID-19, including pneumonia and acute respiratory distress syndrome (ARDS), are well recognized.11 In addition, COVID-19 is associated with deleterious effects on many other organ systems. Common extrapulmonary manifestations of COVID-19 are summarized below.11
The Chinese Center for Disease Control and Prevention published a large case series of COVID-19 in mainland China (72,314 cases, updated through February 11, 2020) and reported:12
- Most patients showed signs of disease after an incubation period of 1–14 days (most commonly around 5 days)1
- Dyspnea and pneumonia developed within a median time of 8 days from illness onset1
SARS-CoV-2 infection may be asymptomatic or it may cause a wide spectrum of symptoms, such as mild symptoms of URT infection and life-threatening sepsis.2 The spectrum of disease reported in 44,672 patients with COVID-19 in China are as follows:12
- Mild: 81% (36,160 cases)
- Severe: 14% (6,168 cases)
- Critical: 5% (2,087 cases)
The estimated prevalence of COVID-19
Since the beginning of the COVID-19 pandemic, daily counts of confirmed cases and deaths have been publicly reported in real-time to control the virus spread.13 However, substantial undocumented infections have obscured the actual fraction of people infected at least once.13 As such, counts of confirmed COVID-19 cases, deaths, and recoveries are insufficient to calculate the number of currently infected individuals.13 The global prevalence of COVID-19 is reported by the World Health Organization (WHO).14
Determining a diagnosis of COVID-19
The first step in managing COVID-19 is the rapid and accurate detection of SARS-CoV-2 enabled by real-time reverse transcription-polymerase chain reaction (RT-PCR).2,15 Nasal swabs, clinical, laboratory, and imaging findings may also be used, as false-negative rates are associated with PCR testing.2
Viral load in respiratory samples climbs in the initial stages of the disease, reaching a peak in the second week, after which viral loads are lowered.15,16
In cases of severe infection, the respiratory fluid virus is highest in the third and fourth weeks.15 Moreover, in patients with comorbidities, the virus persists.15 Positive results can be shown for up to 50 days in disease-recovered individuals.15
Current diagnostic tests for COVID-19 use nucleic acid protein and antibody detection, however, the gold standard remains viral nucleic acid detection by RT-PCR.15 Overall, nucleic acid tests show improved sensitivity and specificity for viral detection over serological test methods.15
Despite the accuracy of RT-PCR, results have not been sufficient to contain rates of viral infection.16 Limitations of RT-PCR include:15
- Viral RNA sequence variation
- Viral evolution
- Inconsistencies in sample storage
- Low-quality nucleic acid purification
- Wait times
Of the nucleic acid-based testing approaches, the most definitive method for the virus is high-throughput sequencing, but this approach is limited due to the cost, equipment, and skillsets required.15
Simplified RT-PCR can also detect diverse regions of the SARS-CoV-2 genome.17 These tests detect the RNA-dependent S and RNA polymerase (RdRp)/helicase (Hel) proteins and the nucleocapsid (N) genes of SARS-CoV-2.18 The RdRp/Hel assays are highly sensitive means for viral detection.15
Other forms of nucleic acid-based technology include:19
- Reverse transcription loop-mediated isothermal amplification (RT-LAMP): an alternative strategy that allows amplification at a constant temperature and eliminates the need for a thermal cycler. Therefore, several methods based on this principle have been developed:
- Reverse transcription loop-mediated isothermal amplification
- Transcription-mediated amplification (TMA)
- Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays
- Nucleic acid hybridization using microarray tests: rely on the generation of complimentary DNA (cDNA) from viral RNA using reverse transcription and subsequent labeling of cDNA with specific probes. These have proven useful in identifying mutations associated with SARS-CoV and have been used to detect up to 24 single nucleotide polymorphisms (SNP) associated with mutations in the S gene of SARS-CoV with 100% accuracy20
- Amplicon-based metagenomic sequencing
Of 112 currently available molecular assays for detecting SARS-CoV-2:19
- 90% utilize PCR or RT-PCR technologies
- 6% utilize isothermal amplification technologies
- 2% utilize hybridization technologies, and
- 2% utilize CRISPR-based technologies
Detection of SARS-CoV-2 antibodies
Serological studies are an alternative to RT-PCR for SARS-CoV-2 diagnostics.15 Serological testing is defined as an analysis of blood serum or plasma and has been operationally expanded to include testing of saliva, sputum, and other biological fluids for the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. IgM or IgG antibodies are produced in response to specific viral antigens including, but not exclusively, the S1/2 subunit, RBD, and nucleocapsid proteins.19 The various forms of serological test include:19
- Enzyme-linked immunosorbent assay (ELISA): a microwell plate-based assay technique for detecting and quantifying biomolecules such as antibodies. The test can be qualitative or quantitative and the result is typically ready after 1–5 hours
- Lateral flow immunoassay: a form of qualitative rapid diagnostic test (RDT), as the result can be obtained in 10–30 minutes. Fluid samples are applied to a substrate material that allows the sample to flow past the band immobilized viral antigens and, if present, anti-CoV antibodies are collected at the band
- Neutralization assay: determines the ability of an antibody to inhibit virus infection of cultured cells and the resulting cytopathic effects of viral replication
- Luminescent immunoassay: involves chemiluminescence or fluorescence
- Biosensor test: converts the specific interaction of antibodies and antigens into a measurable readout via electrical, enzymatic, optical, and other methods
- Rapid antigen test: these tests rely on specific monoclonal antibodies to provide a mechanism for the capture of viral antigens from an analytical sample
What is the prognosis of COVID-19?
Several factors are influencing the prognosis of patients with COVID-19.21
Post-acute COVID-19 syndrome
As the population of patients recovering from COVID-19 increases, an understanding of the healthcare issues concerning these patients is essential.22 Early reports suggest residual effects of SARS-CoV-2 infection, such as dyspnea, fatigue, cognitive disturbances, chest pain, arthralgia, and decline in quality of life.23,24 These multi-organ sequalae are increasingly being appreciated as data and clinical experience in this timeframe accrue.22
As observed in other post-acute viral syndromes described in survivors of other virulent coronavirus epidemics, there are growing reports of prolonged effects after acute COVID-19.22 Patient advocacy groups have informed greater understanding of post-acute COVID-19, a syndrome characterized by persistent symptoms and/or delayed or long-term complications, that occur >4 weeks from the onset of symptoms.22
Post-hospital discharge care of COVID-19 survivors has been recognized as a major research priority by professional organizations:22
- Algorithms for both severe and mild-to-moderate COVID-19 groups recommend:
- Clinical assessment and chest X-ray in all patients and consideration of pulmonary function tests (PFTs), 6-minute walk tests (6MWT), sputum sampling, and echocardiogram according to clinical judgment at 12 weeks
- Evaluation with high-resolution computed tomography (CT) of the chest, CT pulmonary angiogram or echocardiogram
- For those with severe acute COVID-19 (defined as those who had severe pneumonia, required ICU care, are elderly, or have multiple comorbidities), clinical assessment for respiratory, psychiatric, thromboembolic sequelae, and rehabilitation
To address the unknown aspects of post-COVID-19, several active research studies and questions pertaining to post-acute COVID-19 are ongoing.22 As knowledge of COVID-19 sequelae grows, the healthcare needs for patients with sequelae will require using existing outpatient infrastructure, the development of scalable healthcare models, and integration across disciplines for improved mental and physical health in the long term.22
6MWT, 6-minute walk tests; ACE2, angiotensin-converting enzyme 2; CD, cluster of differentiation; cDNA, complementary deoxyribonucleic acid; COVID-19, coronavirus disease 2019; CoVs, coronaviruses; CRP, C-reactive protein; CT, computed tomography; DMVs, double-membrane vesicles; E, envelope; ER, endoplasmic reticulum; ERGIC, ER–Golgi intermediate compartment; ESR, erythrocyte sedimentation rate; Hel, helicase; H-FABP, heart-type fatty acid-binding protein; ICU, intensive care unit; IgG, immunoglobulin G; IgM, immunoglobulin M; LAMP, loop-mediated isothermal amplification; m, meter; M, membrane; N, nucleocapsid; np, nucleoprotein; nsps, nonstructural proteins; ORF, open reading frame; PFTs, pulmonary function tests; PLR, platelet-to-lymphocyte ratio; pp1a/1b, polyprotein 1a/1b; RdRp, RNA-dependent RNA polymerase; RNA, ribonucleic acid; RT-PCR, reverse transcriptase polymerase chain reaction; S, spike; S1/2, spike 1/2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; sg, subgenomic; TMPRSS2, transmembrane protease serine 2; μm, micrometer; WBC, white blood cell.
- Hu B, Guo H, Zhou P, et al. Nat Rev Microbiol 2021;19:141–154.
- Wiersinga WJ, Rhodes A, Cheng AC, et al. JAMA 2020;324(8):782–793.
- Whittaker GR, Daniel S, Millet JK. Curr Opin Virol 2021;47:113–120.
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- Shang J, Wan Y, Luo C, et al. Proc Natl Acad Sci USA 2020;117:11727–11734.
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